Sarah Paule

Posttranslational Activation of Bone Morphogenetic Protein 2 Is Mediated by Proprotein Convertase 6 during Decidualization for Pregnancy Establishment

Bone morphogenetic proteins (BMPs) require major posttranslational modifications to become biologically active. One such key modification is endoproteolytic cleavage of the initially synthesized nonactive precursor protein to release the mature ligand. Here we show in a physiological context of uterine stromal decidualization that BMP2 cleavage is mediated by proprotein convertase 5/6 (PC6). Decidualization is a uterine remodeling event critical for embryo implantation. Deletion or knockdown of either BMP2 or PC6 inhibits decidualization causing implantation failure and female infertility. In this study we provide biochemical and physiological evidence that PC6 proteolytically activates BMP2. We used freshly isolated primary human endometrial stromal cells and demonstrated that PC6 was the sole member of the PC family significantly up-regulated during decidualization. The precursor form of BMP2 was reduced, whereas its active form was increased during decidualization. Inhibition of PC6 activity inhibited decidualization, and this was accompanied by a total blockade of BMP2 activation. Addition of recombinant active BMP2 partially rescued the decidualization arrest caused by PC6 inhibition. PC6 processed BMP2 at the KREKR(282) downward arrow cleavage site, and mutating this site prevented the cleavage. This study thus demonstrates for the first time that the proteolytic activation and thus bioavailability of BMP2 is controlled by PC6.

Proprotein Convertase 5/6 Is Critical for Embryo Implantation in Women: Regulating Receptivity by Cleaving EBP50, Modulating Ezrin Binding, and Membrane-Cytoskeletal Interactions

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

Proteomic approach identifies alterations in cytoskeletal remodelling proteins during decidualization of human endometrial stromal cells.

Decidualization is a tissue remodelling process within the uterus in preparation for embryo implantation and pregnancy. In this study we isolated primary human endometrial stromal cells and stimulated decidualization with cAMP. We then used 2D- differential in-gel electrophoresis (DIGE) to identify proteins induced by decidualization. Eighty-eight out of 2714 spots were differentially regulated, 18 of which were assigned clear identities by mass spectrometry. Many of these are proteins known to be associated with cell structure and cytoskeletal remodelling. We validated five of these proteins by Western blot and immunohistochemistry on human endometrial tissue. The validated proteins are caldesmon 1, src substrate contactin 8, tropomyosin alpha-4 chain, protein disulfide isomerase 1A, and LIM and SH3 domain protein. With the exception of caldesmon 1, none of the identified proteins have previously been associated with decidualization. This study provides insight into our understanding of decidualization, which is important for successful embryo implantation and establishment of pregnancy.

Cleavage of endometrial α-integrins into their functional forms is mediated by proprotein convertase 5/6

BACKGROUND Proprotein convertases (PCs) post-translationally activate a large number of protein precursors through limited cleavage. PC5/6 (PC6) in the human endometrium is tightly regulated during receptivity for embryo implantation. Integrins are transmembrane glycoproteins, some of which play an important role in the adhesive interactions between the trophoblast (blastocyst) and uterine epithelium at implantation. Integrins require PC cleavage for post-translational modification. We hypothesize that pro-integrin-αs in the endometrial epithelium are post-translationally cleaved by PC6 into functional subunits for the binding of blastocyst and adhesion of extracellular matrix proteins. METHODS AND RESULTS We first used the endometrial epithelial cell line, HEC1A, into which siRNA specific to human PC6 (PC6-siRNA) or scrambled sequence (control) was stably transfected. The specific knockdown was confirmed by real-time RT-PCR. PC6-siRNA cells reduced their capacity to attach to trophoblast spheroids and bind to fibronectin compared with control. Knockdown of PC6 decreased cell surface presentation of functional integrins-α1, α2, α5, αV and αVβ5. Western blot analysis demonstrated that PC6 was responsible for the post-translational cleavage of pro-integrin-α5 and integrin-αV into their heavy and light chains in HEC1A cells. We then isolated primary human endometrial epithelial cells and validated that PC6 mediated the post-translational cleavage of integrin-αs in these cells. CONCLUSIONS This study implicates PC6 as a key regulatory protein essential for the attachment of the blastocyst to the endometrial epithelium through the processing of pro-integrin-αs. Compromised PC6 action reduces the post-translational modification of integrin-αs, thus compromising implantation.

Posttranslational removal of α-dystroglycan N terminus by PC5/6 cleavage is important for uterine preparation for embryo implantation in women

Embryo implantation requires a healthy embryo and a receptive endometrium (inner lining of the uterus); endometrial receptivity acquisition involves considerable epithelial surface remodeling. Dystroglycan (DG), a large cell surface glycoprotein, consists of α‐ and β‐subunits; β‐DG anchors within the plasma membrane whereas α‐DG attaches extracellularly to β‐DG. The glycosylated central α‐DG mediates adhesion, but it is obstructed by its large N terminus (α‐DG‐N); α‐DG‐N removal enables DGs adhesive function. We demonstrate here that full‐length α‐DG in the human endometrial epithelium is a barrier for embryo attachment and that removal of α‐DG‐N by proprotein convertase 5/6 (PC6; a protease critical for implantation) regulates receptivity. This was evidenced by: 1) α‐DG contains a PC6‐cleavage site near α‐DG‐N, and PC6 cleaves a peptide harboring such a site; 2) PC6 knockdown reduces α‐DG‐N removal from endometrial epithelial cell surface and blastocyst adhesion; 3) mutating the PC6‐cleavage site prevents α‐DG‐N removal, causing cell surface retention of full‐length α‐DG and loss of adhesiveness; 4) α‐DG‐N is removed from endometrial tissue in vivo for receptivity and uterine fluid α‐DG‐N reflects tissue removal and receptivity. We thus identified α‐DG‐N removal as an important posttranslational control of endometrial receptivity and uterine fluid α‐DG‐N as a potential biomarker for receptivity in women.—Heng, S., Paule, S. G., Li, Y., Rombauts, L. J., Vollenhoven, B., Salamonsen, L. A., Nie, G. Posttranslational removal of α‐dystroglycan N terminus by PC5/6 cleavage is important for uterine preparation for embryo implantation in women. FASEB J. 29, 4011‐4022 (2015). www.fasebj.org

Combination of hydrogel nanoparticles and proteomics to reveal secreted proteins associated with decidualization of human uterine stromal cells

BackgroundIdentification of secreted proteins of low abundance is often limited by abundant and high molecular weight (MW) proteins. We have optimised a procedure to overcome this limitation.ResultsLow MW proteins in the conditioned media of cultured cells were first captured using dual-size exclusion/affinity hydrogel nanoparticles and their identities were then revealed by proteomics.ConclusionsThis technique enables the analysis of secreted proteins of cultured cells low MW and low abundance.

Cytoskeletal remodelling proteins identified in fetal-maternal interface in pregnant women and rhesus monkeys

The uterus undergoes dramatic remodelling in preparation for embryo implantation and pregnancy establishment. A receptive uterus is pivotal for embryo attachment, implantation and the eventual formation of a hemochorial placenta. We have previously identified by proteomics that tropomyosin alpha-4 chain (TPM4), protein disulfide isomerase A1 (PDIA1) and src substrate cortactin 8 (SRC8) were up regulated in the decidualized stromal cells during the late secretory phase of the menstrual cycle in women. These three proteins are associated with cytoskeletal remodelling. This study determined the localization of these three cytoskeletal proteins in the fetal-maternal interface including the decidual cells in the 1st trimester of pregnancy in women and rhesus monkeys. Immunohistochemical analysis revealed that TPM4, PDIA1 and SRC8 were all expressed by the decidual cells and the wall of the spiral arterioles in pregnant women. Similar expression pattern were also found in the rhesus monkey. In addition, TPM4, PDIA and SRC8 were also localized to the trophoblast cells, further highlighting the importance of these cytoskeletal remodelling proteins in early pregnancy.

Proprotein convertase 5/6 cleaves platelet-derived growth factor A in the human endometrium in preparation for embryo implantation

Establishment of endometrial receptivity is vital for successful embryo implantation. Proprotein convertase 5/6 (referred to as PC6) is up-regulated in the human endometrium specifically at the time of epithelial receptivity. PC6, a serine protease of the proprotein convertase family, plays an important role in converting precursor proteins into their active forms through specific proteolysis. The proform of platelet-derived growth factor A (pro-PDGFA) requires PC cleavage to convert to the active-PDGFA. We investigated the PC6-mediated activation of PDGFA in the human endometrium during the establishment of receptivity. Proteomic analysis identified that the pro-PDGFA was increased in the conditioned medium of HEC1A cells in which PC6 was stably knocked down by small interfering RNA (PC6-siRNA). Western blot analysis demonstrated an accumulation of the pro-PDGFA but a reduction in the active-PDGFA in PC6-siRNA cell lysates and medium compared with control. PC6 cleavage of pro-PDGFA was further confirmed in vitro by incubation of recombinant pro-PDGFA with PC6. Immunohistochemistry revealed cycle-stage-specific localization of the active-PDGFA in the human endometrium. During the non-receptive phase, the active-PDGFA was barely detectable. In contrast, it was localized specifically to the apical surface of the luminal and glandular epithelium in the receptive phase. Furthermore, the active-PDGFA was detected in uterine lavage with levels being significantly higher in the receptive than the non-receptive phase. We thus identified that the secreted PDGFA may serve as a biomarker for endometrial receptivity. This is also the first study demonstrating that the active-PDGFA localizes to the apical surface of the endometrium during receptivity.

Ability of GHTD-amide and analogs to enhance insulin activity through zinc chelation and dispersal of insulin oligomers.

GHTD-amide is a tetrapeptide originally isolated from human urine that has hypoglycemic activity. Insulin occurs in secretory granules of beta cells as zinc-stabilized hexamers and must disperse to monomeric form in order to bind to its receptor. The aim of this study was to identify whether GHTD-amide and an analog called ISF402 (VHTD-amide) reduce blood glucose through enhancement of insulin activity by dispersing oligomers of insulin. Peptides containing the HTD-amide sequence and a free alpha-amino group were optimal at binding Zn(2+) and adopting secondary structure in the presence of Zn(2+). Binding was concentration dependent and resulted in a 1:1 Zn:peptide complex. In vitro the tetrapeptides dispersed hexameric insulin to dimers and monomers. GHTD-amide and ISF402 potentiated the activity of hexameric insulin when co-injected into insulin resistant Zucker rats. Injection of peptides with insulin caused reductions in blood glucose and C-peptide significantly larger than achieved with insulin alone, and serum insulin time profiles were also altered consistent with a reduced clearance or enhanced dispersal of the injected insulin. Insulin potentiation by ISF402 was reduced when lispro insulin, which does not form zinc-stabilized hexamers, was used in place of hexameric zinc insulin. In conclusion, GHTD-amide and ISF402 are zinc binding peptides that disperse hexameric insulin in vitro, and potentiate the activity of hexameric insulin more so than monomeric lispro insulin. These results suggest that dispersal of hexameric insulin through chelation of Zn(2+) contributes to the hypoglycemic activity of these tetrapeptides.

Small Molecule Proprotein Convertase Inhibitors for Inhibition of Embryo Implantation

Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compounds lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug.